Skin is the most frequently injured tissue and it is estimated that 100 million people in developed countries acquire scars each year. Injury and subsequent normal tissue repair results in a broad spectrum of scar types, ranging from fine, barely visible discolored lines to a variety of unsightly scars, including those that become stretched over time. In addition, abnormal scar formation results in pathological scars such as hypertrophic scars that are elevated but remain within the boundaries of the original lesion, and keloid scars that are elevated and spread beyond the margins of the original wound. Whether the result of normal or excessive dermal fibrosis, clinical experience shows that patients want less noticeable scars, with color and texture that closely resemble their normal skin.
Scarring is a major cause of physical and psychological morbidity. Therefore, it could be expected that patients undergoing surgical procedures are likely to be highly sensitive to scarring outcomes. Indeed, the high level of dissatisfaction with scarring outcomes among patients is reflected by the high number of patients who undergo scar revision surgery, estimated to be over 150,000 per annum in the USA. In a study published in 2009, a total of 91 percent of patients surveyed agreed that even a small improvement in scarring on a visible body site would be worthwhile. A total of 87 percent agreed that they would consider valuable any adjunct to good surgical technique to prevent or reduce scarring, with 75% of respondents agreeing that they would go to any length to minimize scarring even if the outcome resulted in small improvements in scar appearance. This data confirms that patients are highly concerned about scar appearance following surgery irrespective of age, gender, ethnic background, or geographic location.
A number of different approaches have been used in an effort to manage scarring post-surgery. These range from noninvasive techniques such as silicone gel sheeting, pressure garments, hydrating creams, and ointments to invasive techniques such as steroid injections, lasers, dermabrasion, surgery and even radiation. Unfortunately, many of these approaches are uncomfortable or burdensome for patients and many require a high level of patient compliance. Moreover, these therapies have not been proven to provide entirely satisfactory and reliable results. An increased level of understanding of the processes involved in scarring at the molecular, cellular, and tissue levels will facilitate the development of new pharmaceutical approaches to prevent or treat scarring.
Much effort has gone into identifying factors that may play a causative role in scar formation. In particular, Transforming Growth Factor beta (TGFβ) has been shown to play a role in determining the scarring outcome. TGFβ is a cytokine that has widespread effects on several aspects of growth and development, including controlling normal cellular proliferation, cellular differentiation, and other functions in many cells. The TGFβ-family of proteins includes three isoforms: TGFβ1, TGFβ2, and TGFβ3. Of these, TGFβ1 and TGFβ2 are believed to promote angiogenesis, upregulate collagen production, inhibit collagen degradation, and promote chemo-attraction of inflammatory cells; TGFβ3 is believed to be involved in cell differentiation, embryogenesis and development.
TGFβ is secreted by most cells involved in wound healing, including neutrophils, lymphocytes, macrophages, keratinocytes, dendritic cells, and fibroblasts. Research indicates that once skin is wounded, TGFβ levels rise in response to the injury and can increase the rate of healing and the breaking strength of the repaired tissue. TGFβ also enhances angiogenesis and consequent blood flow to dermal wounds, partly by stimulating the local release of other growth factors.
Some studies suggest that TGFβ1 elicits the rapid movement of neutrophils and monocytes to a wound site. In addition, in vivo animal studies suggest that exogenous TGFβ1 can increase granulation tissue, collagen formation, and wound tensile strength when applied locally or given systemically.
While increased levels of certain TGFβ isoforms are associated with normal reparative processes, increased levels of these proteins are also associated with the formation of excessive fibrous tissue, or fibrogenesis. Indeed, while TGFβ may be involved in normal wound healing and its resultant “normal” scar, hyper-expression of some TGFβ isoforms can result in overproduction of collagen fibers which in turn produces pathological scarring in the form of hypertrophic and keloid scars. Border, W. and Noble, N. (1994) New England Journal of Medicine 331: 1286-1292. For example, in rats, TGFβ1 and TGFβ2 levels are elevated in adult wounds that heal with scar formation, and this scarring can be reduced by using antibodies to inhibit TGFβ1 or TGFβ2 or by addition of TGFβ3. In addition, the addition of TGFβ1 to wounds in rat fetuses (which typically heal without a scar) can cause scar formation. Shah M et al. (1994) J Cell Sci. 107(Pt5):1137-1157.
Neurotoxin prepared from Clostridium botulinum bacterium, and particularly botulinum neurotoxin toxin A (BTX-A), is currently clinically used as a means of temporarily treating the underlying muscle-related factors associated with abnormal muscular spasms of the face, neck, around the eye, and for treating facial lines. BTX-A when injected into specific muscle groups around the face, causes temporary paralysis of these muscles, and thus helps decrease the spasms, as well as the appearance of the lines. In addition, recent studies have shown that deep intradermal injections of BTX-A can act on sweat glands and blood vessels.
The effects of botulinum toxin on TGFβ and wound healing have been studied. For example, studies employing a rat surgical wound model suggest that BTX-A treated wounds induce a muscular paralysis which results in a reduction in inflammation characterized by a reduction in the expression of TGFβ1 in the BTX-A treated wound. Lee, et al. Clinical and Experimental Otorhinolaryngology, Vol. 2, No. 1: 20-27, March 2009. Specifically, the authors hypothesize that the BTX-A induced muscular paralysis sub-adjacent to the wound minimizes repetitive tensile forces on the wound edges, and decreases the inflammatory and fibroblastic response, thus causing a reduction in fibrosis of the wound. The reduced expression of TGFβ1 and decreased inflammatory response at the wound site of the BTX-A group was believed due to a decrease in micro-trauma resulting from the immobilization of the underlying muscle.
Additional studies teach that scar cosmesis may be achieved by immobilizing healing wounds using botulinum toxin. For example, Gassner et al. promotes the use of intramuscular injections of a chemodenervating agent such as botulinum toxin sub-adjacent to a wound to paralyze muscles capable of exerting tension on the wound during the healing process. See Gassner et al., Plast. Reconstr. Surg. 2000 May; 105(6):1948-5; U.S. Pat. No. 6,447,787.
US2006/0067950 also teaches a method of using neurotoxins for wound healing and preventing scar formation by immobilizing the area around the injured tissue by paralyzing the muscles acting on the tissue.
The possible beneficial effects of BTX-A on pre-existing, pathological hypertrophic scars has also been studied with in vitro cultures of fibroblasts isolated from hypertrophic scars. See, e.g., Xiao et al. (2009) Aesthetic Plast Surg 33:409-412; Xiao et al. (2010) Aesthetic Plast Surg 34:424-427; Xiao et al. (2011) Aesthetic Plast Surg 35:802-807. These studies report that BTX-A can inhibit the in vitro growth of fibroblasts from hypertrophic scars and reduce the expression of TGFβ1 and connective tissue growth facton expression in these cells. The authors suggest that BTX-A may have a clinical benefit in the treatment of pre-existing pathological, hypertrophic scars, however, the mechanism of action has yet to be fully appreciated.
The treatment of existing pathological keloid scars using botulinum toxin has also been postulated, but consistent results remain to be seen. U.S. Pat. No. 8,530,410 describes the administration of botulinum toxin to treat “melanin related afflictions” and “skin pigment disorders” such as keloid scars. The patent claims treating a keloid by administering botulinum toxin to the keloid using an amount less than the amount needed to paralyze a muscle. The patent also claims methods for treating a symptom associated with a keloid (i.e., pain, inflammation and vascularization) by intradermal administration of an amount of botulinum toxin less than the amount needed to paralyze a muscle. Treatment of existing dermatofibromas using intra-lesion injections and topical application of BTX-A is also disclosed in this reference. In contrast, other studies, however, teach that intralesional administration of botulinum toxin does not result in regression of keloid tissue, and does not affect the expression of extracellular matrix markers. Gauglitz et al. (2012) Skin Pharmacol Physiol 25:313-318.
Experiments in tissue repair and treatment of existing pathological scars and symptoms associated with such scarring notwithstanding, clinical success has remained elusive, and the various biological and molecular mechanisms responsible for normal tissue repair and pathological scarring have yet to be fully elucidated. Indeed, studies suggest that a potential therapeutic agent may be more or less effective depending on the type and nature of the original wound, thus a “one size fits all” clinical solution is unlikely. See, e.g., Baker et al., Dermatology Research and Practice, Vol. 2009, Article ID 625376; Gold et al. (2014) Dermatol Surg 40:817-824; Gold et al. (2014) Dermatol Surg 40:825-831. As a result, to date there is no approved pharmaceutical product in the US or the EU indicated for the reduction, improvement, or prevention of cutaneous scarring in humans. Thus, unfortunately, scar improvement and prevention of scar formation still remains an area of clearly unmet medical need.
In addition, while prophylactic measures to reduce the incidence and severity of scarring during tissue repair is desirable, there is also a need for additional clinical strategies to promote wound healing in a subject that more closely resembles normal tissue regeneration and thus facilitates the more perfect reconstruction of a wound. To date, attempts at normalization of cutaneous wound repair by promoting a regenerative healing process in injured skin (over tissue repair) include engineering the use of complex biodegradable scaffolds designed to resemble the extracellular matrix of normal skin. While such attempts have met with some success, the need for additional prophylactic methods for promoting a wound healing process in skin that involves less scarring and more tissue regeneration still exists.